In previous studies, we performed a comprehensive analysis of CDK4 as an important target of the 12q13-q14 amplification event in fusion-positive rhabdomyosarcoma (RMS). We hypothesize that there may be additional critical targets within this amplicon. To extend our studies of 12q13-q14 amplification in fusion-positive rhabdomyosarcoma (RMS), we examined Illumina Omni 2.5M array data from a new cohort of 44 fusion-positive RMS cases. Amplification of the 12q13-q14 region was detected in 9 of 28 (32%) PAX3-FOXO1 (P3F)-positive cases, and 1 of 13 (8%) PAX7-FOXO1-positive cases, confirming the preferential occurrence of this amplicon in P3F-positive cases. We focused on the P3F-positive cases and used the Mann-Witney U-test with Bonferroni correction to localize the core region of 12q13-q14 amplification to a 0.74 Mb region containing 41 genes (including 37 protein-coding genes). To identify effector genes expressed at increased levels in 12q13-q14-amplified cases, we performed a similar statistical approach on RNA sequencing data from 5 cases with and 12 cases without 12q13-q14 amplification. This analysis identified 12 differentially expressed genes (including CDK4, OS9 and TSPAN31) within the core-amplified region; these genes showed 4-fold or higher change in expression between amplified and non-amplified cases. These findings in RMS will next be compared with similar analyses of 12q13-q14 amplification and over-expression in other cancer categories, such as glioblastoma and lung adenocarcinoma. In preparation for functional studies of potential amplification targets, we assessed the expression of selected targets in a panel of RMS cell lines. In particular, we assessed the effect of 12q13-q14 amplification on OS9 and TSPAN31 expression in these cell lines. RT-PCR analysis showed that OS9 and TSPAN31 mRNA are expressed at 3-5-fold higher levels in the 12q13-q14-amplified Rh30 cell line compared to P3F-positive lines without 12q13-q14 amplification. We also identified commercial antibodies to initiate Western blot studies of protein expression. OS9 protein expression was detected at higher levels in Rh30 cells compared to P3F-positive lines without 12q13-q14 amplification. In contrast, TSPAN31 protein was expressed at similar or higher levels in Rh30 compared to non-amplified P3F-positive lines. Therefore, 12q13-q14 amplification is associated with high expression of both proteins though there may be copy number-independent overexpression mechanisms in some lines without this amplicon. Additional genes from the 12q13-q14 amplicon will be similarly assessed in RMS cell lines, before commencing functional studies of selected targets in these cell lines. Our previous studies of the 2p24 amplification event in fusion-positive RMS tumors indicated that MYCN was amplified and over-expressed in association with this amplicon. The functional significance of high MYCN expression in fusion-positive RMS was demonstrated in our studies of the gene fusions in RMS in which we show that the combination of MYCN and PAX3-FOXO1 stimulated oncogenic transformation and rapid tumorigenesis of human myoblasts, though neither high MYCN nor high PAX3-FOXO1 expression were sufficient to cause these oncogenic effects. In our first set of experiments, MYCN was constitutively expressed in the human myoblasts. To explore the effect of different levels of MYCN expression and to modulate MYCN expression during experiments, we designed an inducible MYCN expression construct. In particular, the MYCN cDNA was cloned into a lentiviral doxycycline-inducible expression construct, and a hygromycin resistance gene was also introduced in place of the puromycin resistance gene. The resulting construct showed doxycycline induction of high levels of MYCN mRNA and protein in transduced myoblasts. However, in the absence of doxycycline induction, these cells showed significantly higher MYCN expression than cells transduced with the empty expression construct. This low level of leakiness was sufficient to permit oncogenic transformation in the presence of PAX3-FOXO1. Additional studies will be performed to identify transduced cells with lower basal levels of MYCN expression.